New cell lines were developed and characterized in order to study the effect of the Fv-1 gene production transfecting viral DNA. Fv-1 restriction was shown to act at a step prior to formation of normal linear (Form III) viral DNA. Cells which do not synthesize DNA either because of prior treatment with Mitomycin C or following serum starvation remain sensitive to transformation with viral DNA, but fail to support viral replication following infection with MuLV virions. It is likely that transfecting MuLV DNA is able to direct viral replication by a pathway other than that employed by DNA synthesized by infectious MuLV virions.